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Analyzing intracellular peptides generated by proteasomes is highly informative to understand the spatiotemporal regulation of protein homeostasis. A large portion of eukaryotic proteins is proteolyzed within the 20S core particle of the 26S holoenzyme, where proteins are cleaved into peptides of varying lengths. A small percentage of these peptides are presented to the immune system as a representation of the proteome content of the cell. Therefore, understanding the rules that govern proteolytic specificity and product diversity is of relevance not only to biochemistry and proteostasis but also to physiology and immunology. One of the greatest challenges is to separate such proteasome-generated peptides from the total intracellular peptidome due to the susceptibility of short unstructured peptides to myriad proteases and peptidases that are activated upon cell lysis. Here, we describe a simple and rapid method to isolate peptides that are closely associated with proteasomes or trapped inside the core particle of proteasomes in eukaryotic cells. This approach termed PTPs, for proteasome-trapped peptides, requires a limited number of cells as starting materials compared to other published methods yet still provides sufficient yields for mass spectrometry-based proteomic analysis. A single sample obtained from cultured mammalian cells allowed the identification of 1000-2000 different PTPs following LC-MS analysis with high-resolution mass spectrometer.

Ubiquitylation is a reversible posttranslational modification that is critical for most, if not all, cellular processes and essential for viability. Ubiquitin conjugates to substrate proteins either as a single moiety (monoubiquitylation) or as polymers composed of ubiquitin molecules linked to each other with various topologies and structures (polyubiquitylation). This contributes to an elaborate ubiquitin code that is decrypted by specific ubiquitin-binding proteins. Indeed, these different types of ubiquitylation have different functional outcomes, notably affecting the stability of the substrate, its interactions, its activity, or its subcellular localization. In this chapter, we describe protocols to determine whether a protein is ubiquitylated, to identify the site that is ubiquitylated, and provide direction to study the topology of the ubiquitin modification, in the yeast Saccharomyces cerevisiae.

Determining the sequence of protein N-termini and their modifications functionally annotates proteins since translation isoforms, posttranslational modifications, and proteolytic truncations direct localization, activity, and the half-life of most proteins. Here we present in detail the steps required to perform our recently described approach we call Terminal Amine Isotopic Labeling of Substrates (TAILS), a combined N-terminomics and protease substrate discovery degradomics platform for the simultaneous quantitative and global analysis of the N-terminome and proteolysis in one MS/MS experiment. By a 3-day procedure with flexible α- and ɛ-amine labeling and blocking options, TAILS removes internal tryptic and C-terminal peptides by binding to a dendritic polyglycerol aldehyde polymer. Therefore, by negative selection, this enriches for both the N-terminal-labeled peptides and all forms of naturally blocked N-terminal peptides. In addition to providing valuable proteome annotation, the simultaneous analysis of the original mature N-terminal peptides enables these peptides to be used for higher confidence protein substrate identification by two or more different and unique peptides. Second, the analysis of the N-terminal peptides forms a statistical classifier to determine valid isotope ratio cutoffs in order to identify with high-confidence protease-generated neo-N-terminal peptides. Third, quantifying the loss of acetylated or cyclized N-terminal peptides that have been cleaved extends overall substrate coverage. Hence, TAILS allows for the global analysis of the N-terminome and determination of cleavage site motifs and substrates for protease including those with unknown or broad specificity.